Although metastatic breast cancer is still an incurable disease, the increasing understanding of the molecular biology of metastatic disease has allowed the development of therapies aimed at specific molecular targets. To elucidate the mechanistic insights of radioresistant breast cancer cells especially the cancer stem cells will provide important information to define effective radiosensitization targets. Instead of the currently believed aerobic glycolysis, we propose to test a paradigm shifting idea that mitochondrial bioenergetics is the major cellular fuel supply for aggressive cancer growth, especially in the radioresistant breast cancer stem cells. A milestone work from the PI's lab identified a fraction of breast cancer stem cells (BCSCs) expressing HER2 (HER2+ BCSCs) in the radiation-surviving breast cancer cells and in the recurrent/metastatic breast tumors. A unique metabolic feature of HER2+ BCSCs is characterized by a shift from the aerobic glycolysis dominant (Warburg effect) to the mitochondrial governed bioenergetics under radiation insult. Such stress-induced reprogramming of mitochondrial energy boost requires the mitochondrial relocation of CDK1 and phosphorylation of a myriad of critical CDK1 targets inside the mitochondria (i.e., STAT3, SIRT3, complex I subunits, MnSOD). Translocation of normally nuclear cell cycle kinase CDK1 into the mitochondria for enhancing the mitochondrial bioenergetics is a pioneer work in the field of mitochondria and has many implications for tumor metabolism. Cells harboring deficient mitochondria-specific CDK1 showed reduced survival after radiation. These results indicate a new metabolic mechanism by which CDK1 acting upon its mitochondrial targets up-regulates mitochondrial energy output for HER2+ BCSCs to survive and adapt to the therapeutic circumstances, causing aggressive and resistant behavior. The central hypothesis is that activation of CDK1 enhances mitochondrial bioenergetics, leading to the increased capacity of DNA repair and aggressive growth of HER2+ BCSCs; blocking CDK1-mediated mitochondrial bioenergetics is an effective approach to re-sensitize breast tumors to radiation. There are Three Specific Aims: Aim 1. Characterize mitochondrial biogenetics in irradiated breast cancer cells. Aim 2. Investigate CDK1-mediated mitochondrial bioenergetics in HER2+ BCSCs. Aim 3. Evaluate radiosensitization of HER2+ BCSCs by blocking CDK1-mediated mitochondrial bioenergetics. The overarching goals of this proposal are to study how CDK1 regulates the reprogramming of mitochondrial bioenergetics causing radioresistance of breast cancer stem cells HER2+ BCSCs. Subsequently, the mitochondrial CDK1 and/or its key mitochondria substrates will be identified as aspecific molecular target for radiosensitization of resistant breast cancer.